1. Field of the Invention
The present invention relates generally to the fields of molecular virology and immunology. More specifically, the present invention relates to viral defective interfering particles and uses thereof.
2. Description of the Related Art
"Incomplete particles" were discovered by von Magnus in 1947 during successive undiluted passages of influenza viruses (von Magnus '47). In general, these incomplete particles contain less than a full-length genome and are replication-defective. They can be rescued by and interfere with the replication of homologous helper viruses.
To date, most defective interfering particles are discovered in laboratory settings (Holland et al., '87 & '91; Dimmock '96). It is not known if defective interfering particles could also exist in natural infection in humans. Furthermore, most defective interfering studies have demonstrated a correlation between genomic deletion and the defective interfering phenotype. Whether deletion is indeed the cause to the defective interfering phenomenon and whether the identified deletion alone is necessary and sufficient for the defective interfering behavior, have never been proven experimentally.
Another important characteristic of these incomplete particles is their ability to enrich their proportion of the total viral yield in mixed infection with wild type and incomplete viruses (Holland '87). Based on these properties, Huang and Baltimore defined these biologically active defective particles as defective interfering (DI) particles and the replication competent homologous virions as standard virus (Huang & Baltimore, '70). Defective interfering particles are wide-spread in many DNA and RNA viruses in bacteria, plants and animals (Holland, '87; Huang & Baltimore, '77). The biological significance of these defective interfering particles remains an important and intriguing issue in virology and evolutionary biology. Defective interfering particles may play a key role in disease progression of chronic infection.
Hepatitis B virus (HBV) is one of the most common infectious agents in humans (approximately 200 million chronic carriers of HBV worldwide) and chronic active hepatitis B infection leads to the development of cirrhosis and liver cancer (Shih et al, '96). HBV infection is the most common cause of death due to viral infections in humans and is behind only malaria as a cause of death from an infectious agent. Every newborn should be vaccinated against HBV. To date, there is no simple, specific and effective therapy for a deadly fulminant hepatitis B infection.
The molecular and cellular mechanism of chronicity and pathogenesis of HBV infection remains to be elucidated. HBV replication in various hepatoma cell lines in tissue culture does not exhibit any apparent cytopathic effect (Sureau et al., '87; Shih et al., '89). It is generally believed that hepatitis and liver damage are due to immune-mediated cytotoxicity (Milich '91; Chisari & Ferrari, '95). HBV core antigen (HBcAg or nucleocapsid protein) has been shown to be a major target of T cell immunity (Mondelli et al., '82, Vento et al., '85; Ferrari et al., '90; Tsai etal., '92).
Immune escape mutations are known to occur within the major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) epitopes (Pircher et al., '90; Phillips et al., '91). Surprisingly, frequent missense mutations of HBcAg were found to coincide with mapped MHC class II-restricted T cell epitopes (Hosono et al., '95; Lee et al., '96; Bozkaya et al., '96). In addition to these missense mutations, a naturally occurring core antigen internal deletion (CID) was found to be geographically ubiquitous in 4 out of 4 asymptomatic HBV carriers (Okamoto et al., '87), 7 out of 11 chronic active hepatitis (Wakita et al., '91), 2 out of 10 hepatocellular carcinoma tissues (HCC) (Hosono et al, '95) and 6 out of 6 HBV-infected immunosuppressed transplantation patients (Gunther et al., '95). More often, these deletions were in-frame, occurring around HBcAg codon 80-130, and varying in size from 18 to 61 amino acids (approximately 10-33% of wild type HBcAg). The amino terminal moiety of HBcAg is responsible for the polymerization of nucleocapsid particles, while the arginine-rich carboxyl terminus of HBcAg is known to be involved in binding of HBV pregenomic RNA and the reverse-transcribed cDNA (Gallina et al., '89; Hatton et al., '92). The internal deletion of HBcAg is located outside the nucleic acid binding domain and its biological functions have been unclear.
Hepatitis B virus was discovered by Blumberg in 1964 and the initial reports of core internal deletion (CID) mutants of HBV was by Okamoto et al. in 1987 and Wakita et al. in 1991. However, there has been no report of HBV defective interfering particles. Previously, Gerin et al. reported the identification of HBV defective interfering particles based on the morphology of "empty" particles under electron microscope. (American J. of Path., 81:651-668, 1975). As mentioned earlier, the definition of defective interfering is a functional one, not strutural. Morphological features are neither a necessary nor a sufficient criterion for the definition of defective interfering particles. To the contrary, defective interfering particles are not "empty" or without viral genome. Defective interfering particles do have a functionally defective genome. Therefore, defective interfering particles are a life form which can perpetuate itself, while "empty" particles are not life form, since they are simply protein aggregates and do not have a genome to duplicate themselves. Although there has been speculation that CID mutants of HBV could be defective interfering particles (Akarca & Lok, '95), no experimental data, evidence and proof of the four major characteristic features of defective interfering particles, i.e., replication defective, rescuability by helper viruses, interference of helper virus, and enrichment of defective interfering particles, has been reported. As admitted by the authors, "We acknowledge that we do not have direct proof that the deletions result in defective genomes" (p. 1825 near the end, Akarca & Lok, '95). This deficiency in the prior art's ability to determine defective interfering particles is in part due to both technical and conceptual difficulties.
The conventional approach of identifying defective interfering particles relies on plaque assay and infection assay. Since HBV infection in tissue culture is not a well established procedure, and HBV replication in tissue culture does not produce plaques, there is no prior art as to how to determine the presence of defective interfering particles without infection and plaque assay. The CID mutants also contain a number of mutations elsewhere in the HBV genome. It is not obvious how one could circumvent these complications to study the native naturally occurring CID mutation without the enormous complication from other coexisting mutations in the CID variants.
A misconceptual difficulty in the prior art involves the nomenclature of defective interfering particles. In fact, "enrichment" is another equally important feature of defective interfering viruses. The prior art nomenclature often leads to the misconception that the overall viral titer will be dramatically decreased due to a dominant negative effect of defective interfering particles.
Laboratory-derived defective interfering particles of human hepatitis A virus (HAV) have been reported (Siegl et al., '90 and '93). However, these HAV-defective interfering viruses were originated from tissue culture in the laboratory setting. As admitted by the authors (Siegl et al., '90, page 106): " . . . Under conditions of natural infection, however, defective interfering particles of the very same viruses have not yet been observed. Hence, it is not known whether the predicted positive and/or negative effects of this specific class of particles play the expected role during natural infection . . . ". Second, HAV and HBV are different viruses. HAV is an RNA virus transmitted via the oral-fecal route while HBV is a DNA virus transmitted through blood and intimate contact.
The prior art is deficient in the lack of the understanding, experimental support, evidence and proof of the functional behaviors of defective interfering particles in human viruses in natural infections. The present invention fulfills this longstanding need and desire in the art by providing experimental data, evidence and proof of the functional features of human defective interfering viruses.